The long term objective of this grant proposal is to delineate the control of the switching from fetal to adult globin formation during human development. To accomplish this we will apply a somatic cell hybridization approach we have developed which allows us to do observations on the expression of normal, mutant or engineered globin genes in their normal chromosomal location. Using this system, we will investigate first, the mechanism of gamma to beta switching during ontogeny. We will attempt to obtain definitive evidence that globin gene switching is controlled in trans; we will perform studies aiming to detect the physical existence of the postulated development clock of switching and we will attempt to map its chromosomal location. Using fetal or adult hybrids we will investigate the correlation between beta locus replication and the expression of fetal or adult globin genes. Second, we will investigate the control of the beta locus using hybrids containing human beta locus developmental mutants. These studies will include analyses of the functional effects of deletions of the Locus Activation Region; chromatin studies of deletion HPFH and delta beta thalassemia mutants; investigations of genetic heterogeneity and chromosomal mapping of non-deletion HPFH mutants. The proposed studies cannot be done with other experimental systems and are based on the fact that hybrids containing mutant loci can serve as surrogate cells of human erythroid cells. Third, we will use the hybrids as a model system for studying the control of the beta globin locus using homologous recombination. Questions such as the relationship between physical order of genes and order of developmental expression will be asked. Fourth, we will use subtraction hybridization of cDNA libraries from hybrids expressing the fetal or the adult globin program in order to clone genes coding for trans acting factors involved in globin gene switching. Fifth, we will continue our research on human erythroleukemia lines and we will use these lines as well as hybrids in order to study the developmental control of other than globin erythroid characters. Successful completion of the proposed studies will provide several insights on the control of globin genes during development and differentiation.